ABSTRACT Innate-like B cell-derived natural antibodies (NAbs) against T-independent polysaccharide (PS) and phospholipid epitopes are universally present in mammals and serve important roles in the protection against many bacterial pathogens. However robust antibody responses against PS antigens following infection or immunization are absent in neonates. We previously showed that neonatal exposure to PS and phospholipid antigens by immunization or natural colonization with the microbiota, influences the clonal distribution of antigen-specific innate-like B cells leading to quantitative increases in natural antibody (Nab) levels. These effects impact adult susceptibility to infection, and immune-responsiveness to allergens and auto antigens bearing similar B cell epitopes. Although the microbiota impacts the development of other lymphoid lineages its role in the development of the adult innate-like B cell and NAb repertoires is largely unexplored. Our hypothesis is that in addition to the well-described selection processes that occur during B cell development, the accessibility to autologous antigens and exogenous commensal-derived epitopes combine during neonatal development to shape the natural repertoire. The goal of these proposed studies is to elucidate mechanisms of exogenous antigen-directed development of the innate-like B cell repertoire using three model B cell antigen- specificities that represent epitopes for relevant pathogenic and commensal organisms with a spectrum of auto antigenic potential: phosphorylcholine (PC), N-acetyl-D-glucosamine (GlcNAc), and dextran (DEX). In Aim 1 of these studies wild-type gnotobiotic mice will be colonized with defined microbiota to assess the contribution of exogenous antigen signaling on clonal B cell development and formation of the NAb repertoire. Bone marrow chimera systems, composed of antigen-specific immunoglobulin (Ig) heavy chain-transgenic mouse strains, and lineage tracking will enable assessment of endogenous and exogenous antigen signaling on B lymphocyte selection and receptor editing processes. In Aim 2 we will analyze the frequency, cell surface phenotype, subset and isotype distribution as well as immunoglobulin gene usage of human B cells specific for these antigens. Examination of B cells isolated from cord blood, tonsils/adenoids, and peripheral blood B cells of donors of different ages will provide novel insight into the role of antigen experience in the formation of innate- like B cell repertoire diversity within and across individuals. Antigen-specific Ig genes will be cloned and expressed as recombinant antibody to determine effects of antigen-driven maturation on functions of these human antibodies. Collectively, these studies will define the microbial influences on NAb development in mice, determine developmental kinetics of human NAb development, and compare and contrast the NAb repertoire development between both species. This insight into the human infant responses to infection and colonization will advance our long-term goal of developing effective neonatal vaccine strategies and interventional therapies that provide protection from infectious pathogens and allergic and autoimmune diseases.